This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The basic objective of this research continues to be understanding the molecular mechanisms that govern variant antigen gene expression in Plasmodium. Antigenic variation is a fundamental adaptation to evade a host protective immune response and is one of the major factors contributing to the establishment of chronic blood infections. The classic P. knowlesi-rhesus monkey model of malaria has been the primary focus of investigations. This simian malaria model is amenable to both in vitro and in vivo studies and unique stable clones of the P. knowlesi H strain expressing distinct SICA (Schizont Infected Cell Agglutination) variant antigen phenotypes after induced sequential switchings can be maintained after numerous in vivo passages (60 generations) in naive rhesus monkeys. These isogenic clonal lines provide a special tool for studies of the cellular and genetic mechanisms underlying clonal antigenic variation. We hypothesized that studies of the clonal phenotypes provide insights towards understanding the regulation of antigenic variation in vivo. Recent studies have focused on continued genome wide analyses of the large SICAvar gene family, and while the P. knowlesi genome has been published (Pain et al;below), much refinement of the SICAvar data is still needed and this has been ongoing in this laboratory. In the past year, LC-MS/MS analyses of infected erythrocytes has also provided the phenotypic profile of the Pk1(A+)1+, Pk1(B+)1+ and Pk1(C+)1+ clones. Lastly, experiments are underway with collaborators to develop a comprehensive blood-stage transcriptome from RNA samples from an in vivo infection and in vitro adapted cultured parasites.